Molecular Pathophysiology of Congenital Long QT Syndrome.

Ion channels represent the molecular entities that give rise to the cardiac action potential, the fundamental cellular electrical event in the heart. The concerted function of these channels leads to normal cyclical excitation and resultant contraction of cardiac muscle. Research into cardiac ion channel regulation and mutations that underlie disease pathogenesis has greatly enhanced our knowledge of the causes and clinical management of cardiac arrhythmia. Here we review the molecular determinants, pathogenesis, and pharmacology of congenital Long QT Syndrome. We examine mechanisms of dysfunction associated with three critical cardiac currents that comprise the majority of congenital Long QT Syndrome cases: 1) IKs, the slow delayed rectifier current; 2) IKr, the rapid delayed rectifier current; and 3) INa, the voltage-dependent sodium current. Less common subtypes of congenital Long QT Syndrome affect other cardiac ionic currents that contribute to the dynamic nature of cardiac electrophysiology. Through the study of mutations that cause congenital Long QT Syndrome, the scientific community has advanced understanding of ion channel structure-function relationships, physiology, and pharmacological response to clinically employed and experimental pharmacological agents. Our understanding of congenital Long QT Syndrome continues to evolve rapidly and with great benefits: genotype-driven clinical management of the disease has improved patient care as precision medicine becomes even more a reality.

Reevaluation of the requirement for TIP47 in human immunodeficiency virus type 1 envelope glycoprotein incorporation.

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.

Self-perception of health and physical activity levels among the youth and adults before and amidst the COVID-19 pandemic.

Background: Emerging research indicates that the COVID-19 pandemic and associated restrictions led to decreased physical activity levels and poorer health globally. However, most studies on this topic have focused on advanced countries, leaving a gap in understanding the impact in countries like Ghana. This study aimed to fill this gap by assessing self-perceived health status and physical activity levels among youth and adults in Ghana before, during, and after the COVID-19 restrictions. Understanding these dynamics is crucial for informing public health interventions and policies to promote well-being during and beyond the pandemic. Methods: A cross-sectional survey using online data collection methods was conducted, involving 937 participants. Data included demographic information, and International Physical Activity Questionnaire-Short Form (IPAQ-SF). Analysis was done using SPSS version 25, with descriptive statistics and multinomial regression. Results: Most participants (89.6%) reported good health. Male participants were significantly more likely to engage in moderate (1.78 times) and high (3.17 times) physical activity during the COVID-19 period compared to females. Conclusion: This study highlights gender disparities in physical activity levels during the pandemic in Ghana. Addressing these disparities and promoting healthier lifestyles, especially during crises, is crucial for general and mental health. Further research should explore socio-demographic factors' role in health behaviors during pandemics.

Attitudes and behaviour toward snakes on the part of Igbo people in southeastern Nigeria.

Snakes play a crucial role in natural ecosystems, providing ecological services to people by decreasing rodent populations which may cause disease transmission and impair agricultural production. Despite these benefits, snakes are historically a target of persecution and negative attitudes across cultures, and many of them are threatened. Understanding the predictors of snake-human conflicts is essential to improve conservation efforts. We investigated the degree to which emotions, myth beliefs, experience with snakes (via exposure, bites, and knowledge of mortality from a snakebite), and education would predict attitudes toward snakes in a sample of southeastern Nigerian people. We further examined whether attitudes would predict intentional killing of snakes. Ordinal regression analyses revealed that fear, disgust, and belief in the myth that snakes are evil were related to low tolerance of snakes. More frequent encounters with snakes and higher education were associated with higher tolerance of snakes. Furthermore, higher tolerance of snakes was associated with a reduced likelihood of intentionally killing snakes, even when controlling for the influence of the other psychological and experiential variables. Wildlife management education interventions may be important to change attitudes and decrease intentional killing of snakes.

A computational model of Purkinje fibre single cell electrophysiology: implications for the long QT syndrome.

Computer modelling has emerged as a particularly useful tool in understanding the physiology and pathophysiology of cardiac tissues. Models of ventricular, atrial and nodal tissue have evolved and include detailed ion channel kinetics and intercellular Ca(2+) handling. Purkinje fibre cells play a central role in the electrophysiology of the heart and in the genesis of cardiac arrhythmias. In this study, a new computational model has been constructed that incorporates the major membrane currents that have been isolated in recent experiments using Purkinje fibre cells. The model, which integrates mathematical models of human ion channels based on detailed biophysical studies of their kinetic and voltage-dependent properties, recapitulates distinct electrophysiological characteristics unique to Purkinje fibre cells compared to neighbouring ventricular myocytes. These characteristics include automaticity, hyperpolarized voltage range of the action potential plateau potential, and prolonged action potential duration. Simulations of selective ion channel blockade reproduce responses to pharmacological challenges characteristic of isolated Purkinje fibres in vitro, and importantly, the model predicts that Purkinje fibre cells are prone to severe arrhythmogenic activity in patients harbouring long QT syndrome 3 but much less so for other common forms of long QT. This new Purkinje cellular model can be a useful tool to study tissue-specific drug interactions and the effects of disease-related ion channel dysfunction on the cardiac conduction system.

Management of survivors of sexual assault within genitourinary medicine.

Survivors of sexual assault can access treatment and care within genitourinary (GU) medicine services by attending walk-in, booked or a dedicated clinic. Haven Whitechapel the local Sexual Assault Referral Centre (SARC) provides a forensic and aftercare service. A team was set up to ensure efficient communication and clear referral pathways between the centres. This service was audited after eight months of joint working. A retrospective case note review of women attending between August and March 2007 was undertaken. Sixty-nine women were referred to the dedicated clinic. Vaginal rape was reported by 80% of the women. Offer of forensic medical examination documented in 71% presenting within the forensic timeframe. Emergency contraception was offered to 75% of the women. HIV-acquisition risk was documented in 70%. Seventy-eight percent had a sexually transmitted infection screen at their first visit. An HIV test was done to 41% of the women; all were found to be negative. Sixty-two percent women attended follow-up. GU medicine staff should receive specific training in sexual assault. We recommend that GU medicine services and SARC work in partnership to improve the care of victims of sexual assault who access general GU medicine services.

Modelling soil erosion response to sustainable landscape management scenarios in the Mo River Basin (Togo, West Africa).

The rural landscapes in Central Togo are experiencing severe land degradation, including soil erosion. However, spatially distributed information has scarcely been produced to identify the effects of landscape pattern dynamics on ecosystem services, especially the soil erosion control. In addition, relevant information for sustainable land and soil conservation is still lacking at watershed level. On this basis, using the LAndscape Management and Planning Tool for the Mo River basin (LAMPT_Mo), we (1) modelled soil erosion patterns in relation with land use/cover change (LUCC), land protection regime, and landforms, and (2) examined the efficiency of landscape redesign options on soil erosion amounts at basin scale. We found that Simulated historical net soil loss (NSL) for the Mo basin were approximately 26, 23, 27, and 44t/ha/yr, for 1972, 1987, 2000, and 2014, respectively. These simulated NSLs were higher than the tolerable soil loss limits for the Tropics. Steep slopes (≥15°), poorly covered lands (croplands and savannas), and riversides (distances ≤100m) are critical areas of sediment sources. The local appraisal of soil loss was in line with the simulated outputs even though quantification was not accounted for when dealing with rural illiterate people. Furthermore, results showed that the examined management measures, such as controlling the identified erosion hotspots through land protective measures, could help reduce the NSL up to 70%, to values closer to the tolerable limits for the Tropics. The model implementation in the basin showed insights for identifying erosion hotspots and targeting soil conservation planning and landscape restoration measures.

The potential impacts of 21st century climatic and population changes on human exposure to the virus vector mosquito Aedes aegypti.

The mosquito Aedes (Ae). aegypti transmits the viruses that cause dengue and chikungunya, two globally-important vector-borne diseases. We investigate how choosing alternate emissions and/or socioeconomic pathways may modulate future human exposure to Ae. aegypti. Occurrence patterns for Ae. aegypti for 2061-2080 are mapped globally using empirically downscaled air temperature and precipitation projections from the Community Earth System Model, for the Representative Concentration Pathway (RCP) 4.5 and 8.5 scenarios. Population growth is quantified using gridded global population projections consistent with two Shared Socioeconomic Pathways (SSPs), SSP3 and SSP5. Change scenarios are compared to a 1950-2000 reference period. A global land area of 56.9 M km2 is climatically suitable for Ae. aegypti during the reference period, and is projected to increase by 8% (RCP4.5) to 13% (RCP8.5) by 2061-2080. The annual average number of people exposed globally to Ae. aegypti for the reference period is 3794 M, a value projected to statistically significantly increase by 298-460 M (8-12%) by 2061-2080 if only climate change is considered, and by 4805-5084 M (127-134%) for SSP3 and 2232-2483 M (59-65%) for SSP5 considering both climate and population change (lower and upper values of each range represent RCP4.5 and RCP8.5 respectively). Thus, taking the lower-emissions RCP4.5 pathway instead of RCP8.5 may mitigate future human exposure to Ae. aegypti globally, but the effect of population growth on exposure will likely be larger. Regionally, Australia, Europe and North America are projected to have the largest percentage increases in human exposure to Ae. aegypti considering only climate change.

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs.

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells.

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

Multidrug resistance is a component of V79 cell resistance to the alkylating agent adozelesin.

Adozelesin is a highly potent alkylating agent which has entered clinical trials based on its unique mechanisms of action and broad-spectrum antitumor activity in vivo. V79 cells resistant to adozelesin (V79/AdoR) were not resistant to the alkylating agent cisplatin but showed the phenotypic and genotypic characteristics of multidrug resistance. Thus V79/AdoR was cross-resistant to several structurally and functionally unrelated drugs, resistance was reversed by verapamil, and the resistant cell line expressed mdr mRNA and p170 glycoprotein. Also, adozelesin uptake and the amount of drug alkylated to DNA was much lower in the resistant cell line as compared to the sensitive parent. However, even with the same amount of drug bound to DNA (10 fmol/micrograms DNA) the survival of V79/S approximately 15% survival) was much lower than that of V79/AdoR (approximately 80%). Therefore the resistance of V79/AdoR cannot be explained solely by the multidrug resistance mechanism (i.e., lower drug uptake and less drug alkylation to DNA), which suggests that multiple mechanisms may account for resistance to adozelesin. V79/AdoR showed different levels of cross-resistance to several adozelesin analogues. The analogues could be divided into 2 groups; those with very low partition coefficients (log P < 2 as compared to 2.74 for adozelesin) had low levels of cross-resistance, whereas analogues with higher partition coefficients (log P > 2.4) were cross-resistant to adozelesin.

K252a, KT5720, KT5926, and U98017 support paclitaxel (taxol)-dependent cells and synergize with paclitaxel.

We have used paclitaxel-dependent Tax 2-4 cells to screen for compounds that have paclitaxel-like functional activity. The indolocarbazole serine/threonine kinase inhibitor K252a and analogues such as KT5926, KT5720, and K252b partially support the growth of the paclitaxel-dependent cells in the absence of paclitaxel. A novel kinase inhibitor of similar structure, U98017, supports the growth of the dependent cells to 48% of that seen with paclitaxel. Used in combination with paclitaxel, these compounds reduce the amount of paclitaxel required for maximum growth of the dependent cells. Isobologram analysis demonstrates that these compounds also act synergistically with paclitaxel to promote toxicity in wild-type Chinese hamster ovary cells. These selected indolocarbazoles may act at sites distinct from that of paclitaxel and may specifically inhibit kinases that contribute to the destabilization of microtubules. Other indolocarbazoles such as staurosporine and rebeccamycin do not support paclitaxel-dependent cell growth. Structurally unrelated serine/threonine kinase inhibitors such as H-9 and H-7 or tyrosine kinase inhibitors such as lavendustin do not support the growth of these cells. These results define a screen for functional paclitaxel analogues and suggest that it may be useful to investigate the possible synergy of selected indolocarbazoles and paclitaxel in vivo.

Hexokinase redistribution in vivo.

Heterogenous stock mice in addition to mice selectively bred to maximally differ in their severity of alcohol withdrawal seizures (withdrawal seizure-resistant (WSR) and withdrawal seizure-prone (WSP] were used to provide evidence in favor of the importance of the rapidly changing distribution of brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) (HK). An ischemic response at 15, 30, 60 and 120 s after killing showed a decreasing cerebellar cytosolic HK concentration of 31%, 15%, 14% and 10% while the cerebral concentrations were 23%, 13%, 13% and 14%, respectively. WSR and WSP mice given an acute i.p. dose of 4 g/kg of alcohol showed opposite HK responses. Cytosolic HK in WSR mice decreased 18.5%, while WSP mice showed an increase of 20.3% over paired saline-injected controls. When ischemia was allowed to proceed in WSP mice following an in vivo alcohol treatment, cytosolic HK decreased in parallel to mice not given alcohol. These data suggest that alcohol can cause an HK redistribution in vivo which could play a role in the differing sensitivities of WSR and WSP mice to alcohol related seizures.

Ethnic differences in women with HIV infection in Britain and Ireland. The study group for the mrc collaborative study of HIV infection in women.

OBJECTIVE: To examine ethnic differences in the socio-epidemiological and clinical characteristics of a cohort of women with HIV infection in Britain and Ireland. DESIGN AND METHODS: Analysis of baseline data (ethnic group, sexual history, likely route of HIV infection, reasons for HIV testing and first AIDS-defining disease) from 400 women with HIV infection recruited into a cohort study from 15 genitourinary medicine/HIV clinics in Britain and Ireland. RESULTS: Sixty-five per cent of women were white and 29% black African. Their median number of lifetime sexual partners was seven and three, respectively (P < 0.001). Ninety-three per cent of black African and 43% of white women were probably infected through sexual intercourse. Injecting drug use was the most likely route of infection in 55% of white women, but none of the black African women. Perceived risk (33%) or investigation of symptoms (26%) were the most common reasons for HIV testing. Seven per cent of white women and 16% of black African women (P < 0.001) had AIDS when HIV infection was diagnosed. The distribution of first AIDS-defining diagnoses differed (P = 0.001) by ethnic group. For white women, the most common disease was Pneumocystis carinii pneumonia; for black African women it was pulmonary tuberculosis. CONCLUSION: There are important differences between black African and white women in sexual history and route of transmission, disease stage at diagnosis and pattern of AIDS-defining diseases.

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1.

We have previously shown that several protein kinases are present in higher activity levels in multidrug resistant cell lines, such as KB-V1. We have now isolated a gene that codes for a putative protein kinase, PKY, of over 130 kDa that is expressed at higher levels in multidrug-resistant cells. RNA from KB-V1 multidrug-resistant cells was reverse-transcribed and amplified by using primers derived from consensus regions of serine threonine kinases and amplified fragments were used to recover overlapping clones from a KB-V1 cDNA library. An open reading frame of 3648 bp of DNA sequence predicting 1215 aa, has been identified. This cDNA hybridizes to a mRNA of about 7 kb which is expressed at high levels in human heart and muscle tissue and overexpressed in drug-resistant KB-V1 and HL60/ADR cells. Because its closest homolog is the yeast serine/threonine kinase, Yak1, we have called this gene PKY. PKY is also related to the protein kinase family that includes Cdks, Gsk-3, and MAPK proline-directed protein kinases. This protein represents the first of its type known in mammals and may be involved in growth control pathways similar to those described for Yak1, as well as possibly playing a role in multidrug resistance.

Regional teaching improvement programs for community-based teachers.

PURPOSE: Community-based clinical teachers provide an important cadre of faculty for medical education. This study was designed to examine the feasibility and value of an American College of Physicians-sponsored regional teaching improvement program for community-based teachers. SUBJECTS AND METHODS: We conducted five regional (Connecticut, New Hampshire/Vermont, New York, Ohio, and Virginia) 1- to 2-day teaching-improvement workshops for 282 faculty (49% community based, 51% university based). The workshops were conducted by regional facilitators trained by the Stanford Faculty Development Program using large group and small group instructional methods to teach participants a framework for analyzing teaching, to increase their repertoire of teaching behaviors, to define personal teaching goals, and to identify the educational needs of their teaching site. Participants used Likert ratings [1 (low) to 5 (high) scale] to assess workshop quality, facilitator effectiveness, and rewards for and barriers to teaching in their clinics. Using retrospective pre- and postintervention ratings, participants also assessed workshop impacts on teacher knowledge, attitudes, and skills. Finally, participants completed open-ended questions to identify recommended changes to improve their clinic as an educational site for students and residents. RESULTS: At all sites, participants evaluated the program as highly useful (4.6 +/- 0.6, mean +/- SD). Participants' ratings indicated that the program had a positive effect on their knowledge of teaching principles (4.0 +/- 0.9), an increase in their teaching ability (P <0.001), and an increase in their sense of integration with their affiliated institution (P <0.001). CONCLUSIONS: Regional training of university and community faculty can be an effective way of promoting the improvement of teaching and the collaboration between community-based teachers and academic centers. National physician organizations and regionally based facilitators can provide important resources for the delivery of such training.

Staurosporine reduces P-glycoprotein expression and modulates multidrug resistance.

We have investigated the effect of staurosporine and other kinase inhibitors on the mRNA and protein levels of the P-glycoprotein (P-gp) in multidrug resistant (MDR) cells. Treatment of human MDR KB-V1 cells with staurosporine for 24 h caused up to a 50% decrease in the amount of P-gp mRNA and protein present. Co-treatment of KB-V1 cells with verapamil, a known reversal agent, plus staurosporine, H-9, or K252a resulted in an enhanced sensitization of cells to vinblastine than with verapamil alone. These findings support a role for protein kinases in the control of multidrug resistance through effects on P-gp levels.

Non-glucocorticoid steroid analogues (21-aminosteroids) sensitize multidrug resistant cells to vinblastine.

Several members of a group of compounds developed to treat stroke and trauma of the central nervous system are shown also to reverse multidrug resistance in human KB-V1 cells. The most potent reversal agents studied are 21-aminosteroid derivatives (lazaroids), tirilazad mesylate (tirilazad, U-74006F) and U-74389F. Tirilazad sensitizes resistant human cells (KB-V1) to killing by vinblastine by 66-fold, but does not change the sensitivity of the nonresistant parental line, KB-3-1, to vinblastine. KB-V1 cell membranes have high levels of P-glycoprotein, a protein that acts as an efflux pump and is thought to be the major cause of multidrug resistance in these cells. Tirilazad inhibits the photoaffinity labeling of P-glycoprotein with [3H]azidopine in KB-V1 cells more effectively than does verapamil, a standard reversal agent. In addition, tirilazad causes the increased accumulation of [3H]vinblastine in multidrug resistant KB-V1 cells. Studies of the resistance reversal potential of related compounds suggest that the complex amine portion of tirilazad is important for its reversal activity, while the steroid portion is less important.

V79 Chinese hamster lung cells resistant to the bis-alkylator bizelesin are multidrug-resistant.

Bizelesin (U-77779) is a highly potent bis-alkylating antitumor agent that is effective against several tumor systems in vitro and in vivo. V79 cells that were 125- to 250-fold resistant to bizelesin developed after constant exposure to gradually increasing concentrations of the drug. Resistant cells exhibited a multidrug-resistant phenotype and genotype as indicated by cross-resistant to several structurally and functionally unrelated drugs, e.g., colchicine, actinomycin D, and Adriamycin, and overexpression of mdr mRNA. Very low levels of cross-resistance to the alkylating agents cisplatin and melphalan were seen. Multidrug-resistant mouse leukemia (P388/Adriamycin-resistant) and human (KB/vinblastine-resistant) cells were also resistant to bizelesin. Bizelesin resistance was unstable and decreased when cells were grown in the absence of the drug. Resistant and sensitive cell lines had similar levels of glutathione, and bizelesin cytotoxicity for resistant cells was not markedly affected by treatment with buthionine sulfoximine. Cross-resistance between bizelesin and several of its analogs is reported.

ENU-induced mutagenesis at a single A: T base pair in transgenic mice containing phi X174.

Transgenic mice containing the bacteriophage phi X174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies. phi X174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair. Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals. Recoveries of phage from mouse tissues are in the range of 1-5 x 10(7) PFU per micrograms mouse DNA; both recovery and mutation are independent of endogenous CpG methylation. Background mutation frequencies are 2-4 x 10(-7) among phage recovered from liver, brain, spleen, and kidney. Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment. At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four. At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 x) and spleen (10 x). These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.